Compositions and methods for the prevention of S. aureus infection

ABSTRACT

The present invention relates to an immunogenic composition comprising at least one Staphylococcus aureus antigen, wherein said antigen is a polypeptide having at least 80% identity with the SdrH-like polypeptide of SEQ ID NO: 8, Nuc of SEQ ID 
     NO: 4, or LukG of SEQ ID NO: 12, an immunotherapeutic composition comprising a polyclonal antibody which selectively binds to at least one of said antigens, and an in vitro method of identifying an antigen conferring protection against disease caused by S. aureus in a subject.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 9, 2022, isnamed 1778945_ST25.txt and is 246,662 bytes in size.

INTRODUCTION

The present invention relates to immunogenic compositions comprisingStaphylococcus aureus antigens. The present invention further relates toimmunogenic compositions for use in conferring protection againstdisease caused by S. aureus in a subject.

S. aureus is a major cause of infection in humans, and is responsiblefor a wide range of pathologies including skin and soft tissueinfections, osteomyelitis, endocarditis, and sepsis. In particular, S.aureus is responsible for a high proportion of infections associatedwith foreign devices (e.g., catheters) and implants (e.g., prosthetics),due to its ability to form a biofilm on the surfaces of these materials.These infections are particularly problematic as they may be chronic orsystemic, in some cases causing prosthetic joint infection, implantfailure, or even death. As an example, retrospective analysis of S.aureus infections in a large hemodialysis center found that the rate ofS. aureus infection in hemodialysis patients was nearly 18% and wasassociated with a 10% mortality rate, with the large majority ofinfections being associated with vascular catheters (Fitzgerald et al.,2011).

While S. aureus infections are typically treated with antibiotictherapies, the emergence of antibiotic resistant S. aureus bacteria,including methicillin-resistant (MRSA) and vancomycin-resistant (VRSA)strains have complicated the use of conventional antibiotics.Furthermore, while vancomycin is currently the gold standard fortreatment of MRSA bacteremia and endocarditis, this antibiotic not idealdue to poor tissue penetration, undesirable side effects, and slowbactericidal activity (Gould, 2008). In the case of orthopedic implants,revision surgery is most often required (Darouiche, 2004). However, thisstrategy is costly and invasive, and is often more technically difficultthan the initial implant surgery, requires more extensive surgery and isassociated with lower quality of life outcomes in subjects.

The development of effective vaccines preventing S. aureus infectionrepresents a promising alternative to current treatment methods, withvarious vaccines against S. aureus currently under evaluation in phaseI, II, or III clinical trials, though no successful phase III trial hasyet been completed. To date, vaccine development has focused mainly onthe S. aureus secreted alpha toxin (Hla) and/or on the capsularpolysaccharide. While a vaccine targeting alpha toxin has been shown tohave a protective effect against infections in which the toxin isresponsible for the majority of the pathogenic effect (e.g., pneumonia,as described in Bubeck and Schneewind, 2008), it is insufficient forpreventing sub-lethal infections (Adlam et al., 1977). Furthermore,given the variability of capsular polysaccharides, and the fact that alarge number of methicillin-resistant S. aureus strains areunencapsulated, the use of capsular polysaccharide alone is of limitedinterest. Various S. aureus proteins taken alone or in combination arealso under evaluation, including the IsdB protein, which was shown toinduce antibodies in subjects having S. aureus infection, although thiswas insufficient to provide protection against future infection (Zormanet al., 2013). Clinical trials have further shown that vaccination withIsdB has no effect when compared to placebo recipients, and in somecases is even deleterious (McNeely et al., 2014).

As current strategies are unsatisfactory, there remains a need forimproved immunogenic compositions comprising S. aureus antigens orpolyclonal antibodies raised against said antigens. In particular, thereis a need for novel immunogenic and immunotherapeutic compositions thatare able to prevent and/or treat S. aureus infection, for examplecomprising antigens which are able to induce protective antibodiesagainst S. aureus infection or such antibodies themselves. There alsoremains a need for novel methods of identifying antigens conferringprotection against disease caused by S. aureus in a subject. Inparticular, in view of the high cost and duration of clinical trialsthere exists a need for an improved assay that may be used as acorrelate of protection for assessing vaccine responses.

The present invention fulfils these and other needs by providing animmunogenic composition, an immunotherapeutic composition, and an invitro method of identifying an antigen conferring protection againstdisease caused by S. aureus in a subject.

In particular, the present invention provides an immunogenic compositioncomprising at least one Staphylococcus aureus antigen, wherein saidantigen is a polypeptide having at least 80% identity with the SdrH-likepolypeptide of SEQ ID NO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO:12.

According to a particular aspect, the immunogenic composition comprisesan antigen having at least 80% identity with the SdrH-like polypeptideof SEQ ID NO: 8 and an antigen having at least 80% identity with LukG ofSEQ ID NO: 12.

The one or more antigens comprised in the immunogenic composition of theinvention advantageously provide unexpected, improved immunogenicproperties (e.g., level, quality and/or scope of the immunogenicresponse) as compared to existing antigens, such as IsdB.

Preferably, the immunogenic composition comprises the S. aureus antigensin the form of separate polypeptides or in the form of one or morefusion polypeptides or both in the form of separate polypeptide(s) andfusion polypeptide(s).

Preferably, the immunogenic composition further comprises apharmaceutically acceptable excipient.

Preferably, the immunogenic composition is for use as a vaccineconferring protection against disease caused by S. aureus in a subject

The present invention further relates to an immunotherapeuticcomposition comprising a polyclonal antibody which selectively binds toat least one antigen as defined herein, wherein said antibody promotesuptake and killing of S. aureus by phagocytes.

Preferably, the immunotherapeutic composition further comprises apharmaceutically acceptable excipient.

Preferably, the immunotherapeutic composition is for use as a passiveimmunotherapy conferring protection against disease caused by S. aureusin a subject.

Preferably, said S. aureus is a methicillin-resistant S. aureus (MRSA)or a methicillin-susceptible S. aureus (MSSA).

Preferably, said subject has an osteoarticular device, preferably anosteoarticular implant, more preferably a total joint replacementprosthesis.

Preferably, said immunogenic or immunotherapeutic composition providedherein is for use in association with one or more antibiotics effectiveagainst a S. aureus infection.

The present invention further relates to an in vitro method ofidentifying an antigen conferring protection against disease caused byS. aureus in a subject comprising:

-   -   a) incubating a solution comprising S. aureus with a solution        comprising antibodies raised against an S. aureus antigen,        preferably for one hour at 35° C., thereby obtaining a mixed        suspension,    -   b) contacting macrophages with the mixed suspension of step a),    -   c) removing the mixed suspension from macrophages and adding        fresh medium supplemented with antibiotics to kill        extracellular S. aureus bacteria, and    -   d) assessing internalization and killing of S. aureus bacteria        by said macrophages, wherein said antigen is considered to        confer protection against disease caused by S. aureus when said        antigen induces both increased internalization and killing of S.        aureus while preserving the viability of macrophages.

In contrast to previous methods, which notably use polymorphonuclearneutrophils, the inventors have developed a novel OPA assay foridentifying target vaccine antigens capable of generating antibodiespromoting both uptake and killing of S. aureus. Indeed,polymorphonuclear neutrophils show very strong bactericidal activity(“killing”), which notably makes it impossible to evaluate whether ornot eventual “facilitating” antibodies (i.e., which promote bacterialuptake but which then result in intracellular bacterial growth ratherthan killing), are generated. Advantageously, macrophages have a muchlower bactericidal (“killing”) activity than polymorphonuclearneutrophils, due to their lower levels of synthesis of reactive oxygenspecies and antimicrobial peptides. Furthermore, in the specific contextof osteoarticular prosthetic infections, the development of amacrophage-based assay is particularly advantageous, as, in thephysiopathology of infection, circulating blood-borne S. aureus must becleared from the bloodstream by macrophages present in the spleen and/orlungs rather than by polymorphonuclear neutrophils, thereby reducing theduration of bacteremia and the probability of establishing a prostheticinfection.

Preferably, said macrophages are an immortalized macrophage cell line,preferably the J774.2 cell line.

Preferably, the killing of S. aureus bacteria in step d) is assessed bycomparing the quantity of bacteria internalized in macrophages 3 hoursafter step c) with the quantity of bacteria internalized in macrophages6 hours after step c).

DESCRIPTION OF THE INVENTION

Before describing the invention in further detail, it should be notedthat the terms “a” and “an” as used herein are used in the sense thatthey mean “at least one”, “at least a first”, “one or more” or “aplurality” of the referenced compounds or steps, unless the contextdictates otherwise. The term “and/or” as used herein includes themeaning of “and”, “or” and “all or any other combination of the elementsconnected by said term”. The term “comprising”, “having”, “including”,or “containing” (and any form of said terms, such as e.g., “contains” or“contain”) are open-ended and do not exclude additional, unrecitedelements or method steps. In contrast, the term “consisting of” as usedherein excludes any other components (beyond trace levels) or steps.

As indicated above, according to a first aspect, the present inventionrelates to an immunogenic composition comprising at least oneStaphylococcus aureus antigen, wherein said antigen is a polypeptidehaving at least 80% identity with the SdrH-like polypeptide of SEQ IDNO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO: 12.

The term “immunogenic” as used herein refers to the ability of thecomposition to induce or stimulate a measurable B cell-mediated immuneresponse in a subject into which the component qualified as immunogenichas been introduced. For example, the composition of the invention isimmunogenic in the sense that it is capable of inducing or stimulatingan immune response in a subject which can be innate and/or specific(i.e., against at least one S. aureus polypeptide comprised in saidimmunogenic composition), humoral and/or cellular (e.g., production ofantibodies and/or cytokines and/or the activation of cytotoxic T cells,B, T lymphocytes, antigen presenting cells, helper T cells, dendriticcells, NK cells, etc). The immunogenic composition usually results in aprotective response in the administered subject. Specifically, thecomposition of the invention is immunogenic in that it inducesantibodies recognizing at least one S. aureus polypeptide and increasesboth the uptake and the killing of S. aureus by phagocytes. However,said composition may also induce one or more additional immuneresponses.

Specifically, the inventors have surprisingly shown here that each ofthe SdrH-like polypeptide, Nuc, and LukG antigens is able to induceantibodies increasing both the uptake and the killing of S. aureus byphagocytes. The generation of antibodies having such activity, performedfor the first time here in a macrophage-based model, is indicative thatthe antigens described herein induce protection against S. aureusinfection when present in an immunogenic composition. Indeed, theresults obtained here, with the antigens of the invention, are innotable contrast with those obtained with IsdB, previously shown to havedeleterious effects (as S. aureus infection may be favored), confirmingthe pertinence of this macrophage-based model in evaluating antigens. Invivo results obtained in an animal model further show that theseantigens are able to reduce S. aureus bacterial growth in the kidneys toa larger extent than that observed with adjuvant alone and/or with acontrol antigen such as staphylokinase, further confirming their abilityto induce protection against S. aureus infection. Thus, according to aparticular aspect, the immunogenic composition comprises at least oneStaphylococcus aureus antigen inducing antibodies against said antigenincreasing both the uptake and the killing of S. aureus uponphagocytosis of the bacteria, wherein said antigen is a polypeptidehaving at least 80% identity with the SdrH-like polypeptide of SEQ IDNO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO: 12.

As used herein, the term “S. aureus antigen” refers to a polypeptidepresent in or obtained from a S. aureus species or a fragment thereof(e.g., an epitope) capable of being bound by an antibody, wherein saidantigen is selected from an “SdrH-like” polypeptide, Nuc, and LukG, andcombinations of one or more thereof. Typically, such an antigen containsone or more B epitope(s). In the context of the invention, this termencompasses native S. aureus antigens (e.g., a full-length antigen) ormodified versions (e.g., fragments or variants) thereof. A “native” S.aureus antigen can notably be found, isolated, obtained from a source ofS. aureus in nature. Such sources include biological samples (e.g.,blood, plasma, sera, saliva, sputum, tissue sections, biopsy specimens,etc.) collected from a subject that has been infected with or exposed toS. aureus, cultured cells, as well as recombinant materials available indepositary institutions (e.g., ATCC or TB institutions), libraries ordescribed in the literature (e.g., S. aureus isolates, S. aureusgenomes, etc.).

The “SdrH-like” antigen or polypeptide is a cell wall-anchoredserine-aspartate repeat family protein containing the host attachmentdomain MSCRAMM (microbial surface components recognizing adhesive matrixmolecules). The “SdrH-like” polypeptide may comprise the sequence of SEQID NO: 7 or 8, which may be encoded by the nucleotide sequence of SEQ IDNO: 5 or 6, respectively. In the context of the present invention, the“SdrH-like” polypeptide preferably has the sequence of SEQ ID NO: 8.

The “Nuc” antigen (also known as micrococcal nuclease or thermonuclease)is an extracellular nuclease. After cleavage by a signal peptidase atthe cell membrane, Nuc may be processed into two active forms: NucA orNucB. Nuc may notably comprise the sequence of SEQ ID NO: 3 or 4, whichmay be encoded by the nucleotide sequence of SEQ ID NO: 1 or 2. In thecontext of the present invention, Nuc preferably has the sequence of SEQID NO: 4.

The “LukG” antigen (also known as LukA) forms a heterodimer with “LukH”(also known as LukB). This heterodimer, LukGH, is a pore-formingleucocidin that at least partially mediates killing of immune cells,such as human monocytes, macrophages, and polymorphonuclear cells by S.aureus. LukG may comprise the sequence of SEQ ID NO: 11 or 12, which maybe encoded by the nucleotide sequence of SEQ ID NO: 9 or 10,respectively. In the context of the present invention, LukG preferablyhas the sequence of SEQ ID NO: 12.

While LukG forms a heterodimer with LukH, preferably said immunogeniccomposition comprises LukG in the absence of LukH. Indeed, the inventorshave surprisingly found that antibodies increasing the uptake and thekilling of S. aureus by phagocytes may be induced by LukG when takenalone (i.e., in the absence of LukH). This is in notable contrast toprevious studies which suggest that the LukGH heterodimer must be usedto generate antibodies. In particular, Badarau et al. 2016 found thatmonoclonal antibodies raised against either LukG or LukH alone had verylittle or even no ability to neutralize the LukGH toxin. Thus, accordingto a preferred aspect, while the immunogenic composition may compriseboth LukG and LukH it preferably comprises LukG in the absence of LukH.

The skilled person will understand that, as a result of the degeneracyof the genetic code, there are many nucleotide sequences that may encodea polypeptide as described herein. In particular, codon usage within agiven nucleotide sequence may be adapted for optimized expression of thecorresponding polypeptide in an organism other than S. aureus (e.g., E.coli).

A modified S. aureus antigen (e.g., a variant) typically differs from apolypeptide specifically disclosed herein or a native polypeptide at oneor more position(s), for example via one or more amino acidsubstitutions, insertions, additions and/or deletions, non-naturalarrangements, and any combination thereof. Amino acid substitutions maybe equivalent or not. Preferably, the substitution is made with an“equivalent” amino acid, i.e., any amino acid whose structure is similarto that of the original amino acid and therefore unlikely to change thebiological activity of the antigen. Examples of such substitutions arepresented in Table 1 below:

TABLE 1 Substitutions with equivalent amino acids Original amino acidSubstitution(s) Ala (A) Val, Gly, Pro Arg (R) Lys, His Asn (N) Gln Asp(D) Glu Cys (C) Ser Gln (Q) Asn Glu (G) Asp Gly (G) Ala His (H) Arg Ile(I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met (M) Leu Phe (F) Tyr Pro(P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) Tyr Tyr (Y) Phe, Trp Val(V) Leu, Ala

When several modifications are contemplated, they may concernconsecutive and/or non-consecutive residues. Modification(s) may begenerated by a number of ways known to the skilled person, such assite-directed mutagenesis, PCR mutagenesis, DNA shuffling and bysynthetic techniques (e.g., resulting in a synthetic nucleic acidmolecule encoding the desired polypeptide variant).

Regardless of the origin of the S. aureus antigen (e.g., native ormodified), the antigen comprised in the immunogenic composition of theinvention retains one or more immunogenic portions of the correspondingnative antigen, more preferably B epitope(s). Methods to identify theappropriate immunogenic portion of an antigen are well-known in the art.

The term “polypeptide” as used herein refers to a polymer of amino acidresidues which comprises at least 10 or more amino acids, preferably atleast 20 or more amino acids, bonded via covalent peptide bonds. Thepolypeptide may be linear, branched or cyclic and may comprise naturallyoccurring and/or amino acid analogs. It may be chemically modified(e.g., being glycosylated, lipidated, acetylated, cleaved, cross-linkedby disulfide bridges and/or phosphorylated). It may comprise additionalelements such as a tag (e.g., his, myc, Flag, etc.) and/or a targetingpeptide (e.g., signal peptide, trans-membrane domain, etc.). Preferably,the at least one polypeptide comprised in the immunogenic composition ofthe present invention does not comprise a signal peptide. Preferably,the at least one polypeptide comprised in the immunogenic composition ofthe invention does not comprise a tag. It will be understood that theterm “polypeptide” encompasses proteins (usually employed forpolypeptides comprising 50 or more amino acid residues), oligopeptides,and peptides (usually employed for polypeptides comprising less than 50amino acid residues). Each polypeptide may thus be characterized byspecific amino acids and be encoded by specific nucleic acid sequences,such as those provided herein.

Thus, a polypeptide “comprises” an amino acid sequence when the aminoacid sequence is a part of the final amino acid sequence of thepolypeptide. Such a polypeptide may in some cases have up to severalhundred additional amino acids residues (e.g., tag peptides, targetingpeptides, etc.). A polypeptide “consists of” an amino acid sequence whenthe polypeptide does not contain any amino acids other than that of therecited amino acid sequence.

The term “percent (%) identity” refers to an amino acid to amino acid ornucleotide to nucleotide correspondence between two polypeptide ornucleic acid molecules. The percentage of identity between two moleculesis a function of the number of identical positions shared by thesequences, taking into account the number of gaps which must beintroduced for optimal alignment and the length of each gap. The percentidentities referred to in the context of the present invention aredetermined after optimal alignment of the sequences to be compared,which may therefore comprise one or more insertions, deletions,truncations and/or substitutions. This percent identity may becalculated by any sequence analysis method well-known to the personskilled in the art. In particular, the percent identity may bedetermined after global alignment of the sequences to be compared of thesequences taken in their entirety over their entire length. In additionto manual comparison, it is possible to determine global alignment usingthe algorithm of Needleman and Wunsch (1970).

For nucleotide sequences, the sequence comparison may be performed usingany software well-known to a person skilled in the art, such as theNeedle software. The parameters used may notably be the following: “Gapopen” equal to 10.0, “Gap extend” equal to 0.5, and the EDNAFULL matrix(NCBI EMBOSS Version NUC4.4).

For amino acid sequences, the sequence comparison may be performed usingany software well-known to a person skilled in the art, such as theNeedle software. The parameters used may notably be the following: “Gapopen” equal to 10.0, “Gap extend” equal to 0.5, and the BLOSUM62 matrix.

Preferably, the percent identify as defined in the context of thepresent invention is determined via the global alignment of sequencescompared over their entire length.

The present invention encompasses polypeptide sequences havingsubstantial sequence identity to the polypeptides disclosed herein,preferably comprising at least 50% sequence identity, preferably atleast 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity with apolypeptide sequence provided herein using the methods described above.According to a preferred embodiment, the polypeptide has at least 80%identity with the SdrH-like polypeptide, Nuc, or LukG of S. aureussubsp. aureus Mu50 (Accession no. BA000017.4). Preferably, thepolypeptide has at least 80% identity with the SdrH-like polypeptide ofSEQ ID NO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO: 12, even morepreferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or higher identity. Preferably, the polypeptidehas 100% identity with the SdrH-like polypeptide of SEQ ID NO: 8, Nuc ofSEQ ID NO: 4, or LukG of SEQ ID NO: 12.

The immunogenic composition provided herein may comprise any combinationof the polypeptides provided herein. As a non-limiting example, thecomposition may comprise a polypeptide having at least 80% identity withNuc of SEQ ID NO: 4 and a polypeptide having at least 80% identity withLukG of SEQ ID NO: 12. Alternatively, the composition may comprise apolypeptide having at least 80% identity with Nuc of SEQ ID NO: 4 and apolypeptide having at least 80% identity with the SdrH-like polypeptideof SEQ ID NO: 8. Alternatively, the composition may comprise apolypeptide having at least 80% identity with the SdrH-like polypeptideof SEQ ID NO: 8 and an antigen having at least 80% identity with LukG ofSEQ ID NO: 12. Alternatively, the composition may comprise a polypeptidehaving at least 80% identity with Nuc of SEQ ID NO: 4, a polypeptidehaving at least 80% identity with LukG of SEQ ID NO: 12, and apolypeptide having at least 80% identity with the SdrH-like polypeptideof SEQ ID NO: 8. Preferably, the immunogenic composition comprises anantigen having at least 80% identity with the SdrH-like polypeptide ofSEQ ID NO: 8 and an antigen having at least 80% identity with LukG ofSEQ ID NO: 12.

The antigens provided herein advantageously induce antibodies increasingboth the uptake and the killing of S. aureus by phagocytes. Thus, theimmunogenic composition of the invention advantageously comprises atleast one S. aureus antigen inducing antibodies that increase both theuptake and the killing of S. aureus by phagocytes, wherein said antigenis a polypeptide having at least 80% identity with the SdrH-likepolypeptide of SEQ ID NO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO:12. Without being limited by theory, the antibody may facilitatephagocytosis or antibody dependent cellular cytotoxicity (ADCC), orboth, of a S. aureus bacterium. In one case, the antigen binding portionof the opsonizing antibody binds to a target antigen, whereas the Fcportion of the opsonizing antibody binds to an Fc receptor on aphagocyte. In other cases, the antigen binding portion of the opsonizingantibody binds to a target antigen, whereas the Fc portion of theopsonizing antibody binds to an immune effector cell, e.g., via its Fcdomain, thus triggering target cell lysis by the bound effector cell(e.g., monocytes, neutrophils and natural killer cells).

The immunogenic composition provided herein may comprise the S. aureusantigens in the form of separate polypeptides or in the form of one ormore fusion polypeptides or both in the form of separate polypeptide(s)and fusion polypeptide(s) when multiple polypeptides are present in theimmunogenic composition. As used herein, the term “fusion polypeptide”means a polypeptide created by joining two or more polypeptide sequencestogether. The fusion polypeptides encompassed in this invention includetranslation products of a chimeric gene construct that joins the DNAsequences encoding one or more antigens, or fragments or mutantsthereof, with the DNA sequence encoding a second polypeptide to form asingle open-reading frame. In other words, a “fusion polypeptide” is arecombinant protein of two or more proteins which are joined by apeptide bond or via several peptides.

The immunogenic composition provided herein may further comprise thesame or different quantities of each component when two or morepolypeptides are comprised in the immunogenic composition. As anon-limiting example, a total quantity of 50 μg of antigen may beadministered per dose. It is appreciated that optimal quantity of saidone or more S. aureus antigens can be determined by the artisan skilledin the art.

A further aspect of the present invention is the immunogenic compositionas provided herein for use as a vaccine conferring protection againstdisease caused by S. aureus in a subject. The composition comprises asufficient quantity of said one or more antigens so as to betherapeutically effective. Preferably, said vaccine is administered to asubject that does not have an existing S. aureus infection so as toinduce a S. aureus-protective humoral or cellular immune response insaid subject. Alternatively, said vaccine may be administered to asubject in which S. aureus infection has already occurred but that is ata sufficiently early stage such that that the immune response producedto the vaccine effectively inhibits further spread of S. aureusinfection. This may notably be the case when S. aureus bacteremia (SAB)occurs, but that has not yet caused more serious infection, such asbloodstream infection or septicemia.

Said immunogenic composition or vaccine may be administered as a singledose. Alternatively, said immunogenic composition or vaccine may beadministered as in multiple doses over a period of time. In particular,administration of the vaccine may be repeated as appropriate to maintainthe protective effect.

Said immunogenic composition or vaccine may further comprise one or moreadjuvants, which serve to enhance the magnitude, quality and/or durationof the immune response. Adjuvants for immunogenic compositions andvaccines are well-known in the art. As a non-limiting example, saidadjuvants include incomplete or complete Freund's adjuvant,monoglycerides and fatty acids (e. g. a mixture of mono-olein, oleicacid, and soybean oil), mineral salts such as aluminum salts (e.g.,aluminum hydroxide, aluminum phosphate, aluminum sulfate) or calciumphosphate gels, oil emulsions and surfactant based formulations (e.g.,MF59 (microfluidised detergent stabilized oil-in-water emulsion), QS21(purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),MPL-SE, Montanide ISA-51 and ISA-720 (stabilised water-in-oilemulsion)), particulate adjuvants (e.g., virosomes (unilamellarliposomal vehicles incorporating influenza haemagglutinin), AS04([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins andlipids), polylactide co-glycolide (PLG)), natural and syntheticmicrobial derivatives (e.g., monophosphoryl lipid A (MPL), Detox (MPL+M.Phlei cell wall skeleton), AGP [RC-529] (synthetic acylatedmonosaccharide), Detox-PC, DC Chol (lipoidal immunostimulators able toself-organize into liposomes), OM-174 (lipid A derivative), CpG motifs(synthetic oligonucleotides containing immunostimulatory CpG motifs),genetically modified bacterial toxins to provide non-toxic adjuvanteffects, such as modified LT and CT), endogenous human immunomodulators(e.g., hGM-CSF or hIL-12 (cytokines that can be administered either asprotein or plasmid encoded), Immudaptin (C3d tandem array), MoGM-CSF,TiterMax-G, CRL- 1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, andMF59) and inert vehicles, such as gold particles. Preferably, theadjuvant is a mineral salt, preferably among those listed above, morepreferably aluminum hydroxide and/or aluminum phosphate. Preferably, theadjuvant is formulated as a wet gel suspension, as is the case for theAlhydrogel® and Adju-Phos® adjuvants commercialized by InvivoGen.Preferably, the ratio of antigen (Ag) to adjuvant is 0.4 to 3 mg Ag:mgaluminum (Al).

In a further aspect, the present invention relates to animmunotherapeutic composition comprising an antibody which selectivelybinds to at least one S. aureus antigen as provided herein (e.g., apolypeptide having at least 80% identify with the SdrH-like polypeptide,Nuc, or LukG), wherein said antibody promotes uptake and killing of S.aureus by phagocytes.

As used herein, the expression “immunotherapeutic composition” refers toa composition that comprises immune molecules (e.g., antibodies and,optionally, additional immune molecules) and that provides passiveimmunity. “Passive immunity” refers more particularly to any immunityconferred to a subject without administration of an antigen. It isgenerally temporary and short term (e.g., providing immunity for weeksor months).

As used herein, the term “antibody” refers to any polypeptide thatcomprises at least an antigen binding fragment or an antigen bindingdomain and that selectively binds a target antigen. Thus, theimmunotherapeutic composition may notably include antibodies orpolypeptides comprising antibody CDR domains that bind to one or more S.aureus antigens. In certain cases, it is understood that antibodybinding to the target antigen is still selective despite some degree ofcross-reactivity. Typically, binding between an antibody and an antigenis considered to be specific when the association constant K_(A) ishigher than 10⁻⁶ M. The antibody comprised in the immunotherapeuticcomposition provided herein may be polyclonal, monoclonal, monospecific,polyspecific, human, humanized, single chain, chimeric, synthetic,recombinant, or any fragment of such an antibody that retains selectiveantigen binding, including, but not limited to, Fab, F(ab′)₂, Fv andscFv fragments. Antibodies may be whole immunoglobulin of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁ and IgA₂) or subclass. Preferably, the antibody providedherein is polyclonal. Thus, according to a preferred embodiment, theimmunotherapeutic composition of the invention comprises a polyclonalantibody which selectively binds to at least one antigen as providedherein, wherein said antibody promotes uptake and killing of S. aureusby phagocytes.

The term “polyclonal antibody” as used herein more particularly refersto a mixture of antibody molecules which are capable of binding to orreacting with several different specific antigenic determinants on thesame or on different antigens. Polyclonal antibodies are thus derivedfrom different B cell lineages. The variability in antigen specificityof a polyclonal antibody is located in the variable regions of theindividual antibodies constituting the polyclonal antibody, inparticular in the complementarity determining regions CDR1, CDR2, andCDR3. The polyclonal antibody may be prepared by immunization of ananimal, such as a horse, cow, bird, rabbit, mouse, or rat with thetarget antigen or portions thereof, by display (e.g., phage, yeast orribosome display) or hybridoma techniques. Polyclonal antibodypreparations may be isolated from the blood, milk, colostrum or eggs ofimmunized animals, and typically include antibodies that are notspecific for the target antigen in addition to antibodies which arespecific for the target antigen. Antibodies specific for the targetantigen may be purified from the polyclonal antibody preparation or thepolyclonal antibody preparation may be used without furtherpurification. Thus, the term “polyclonal antibody” as used herein refersto both antibody preparations in which the antibody specific for thetarget antigen has been enriched and to preparations that are notpurified. The polyclonal antibody may be provided in isolated form, insolution (e.g., animal antisera) or in host cells (e.g., hybridomas).According to a particular aspect, the immunotherapeutic composition maybe a polyclonal antiserum. Numerous techniques are known to those in theart for enriching polyclonal antibodies for antibodies to specificantigens. In a certain aspect, the antibody or antibodies may beaffinity purified from an animal or second subject that has beenchallenged with the antigen(s) provided herein. Recombinant productionof highly specific polyclonal antibodies suitable for prophylactic andtherapeutic administration as provided in WO 2004/061104, incorporatedherein by reference in its entirety, may also be used. Recombinantpolyclonal antibody (rpAb) can be purified from a production bioreactoras a single preparation without separate handling, manufacturing,purification, or characterization of the individual members constitutingthe recombinant polyclonal protein. Alternatively, in some cases, it maybe envisaged that the polyclonal antibody is prepared by mixing multiplemonoclonal antibodies.

The immunotherapeutic compositions of the present invention may be usedfor therapeutic purposes, e.g., for treating a subject after exposure toS. aureus. The immunotherapeutic composition may also be usedprophylactically, prior to an expected or possible exposure to S. aureus(e.g., prior to orthopedic surgery, kidney dialysis). Saidimmunotherapeutic composition may be advantageously used for theprevention or treatment of infection by strains of S. aureus that carrythe corresponding antigen(s) (e.g., “SdrH-like” polypeptide Nuc and/orLukG). Administration may be repeated as necessary to provide passiveimmunity over a given period of time or prior to specific events (e.g.,prior to surgery).

Preferably, said prevention or treatment of infection occurs by passiveimmunization. Thus, according to a preferred embodiment, theimmunotherapeutic composition provided herein is for use as a passiveimmunotherapy conferring protection against disease caused by S. aureusin a subject. In this regard, the immunotherapeutic composition may be apolyclonal composition. In a particular embodiment, theimmunotherapeutic composition is a polyclonal antiserum, preferablyaffinity purified, from an animal which has been challenged with“SdrH-like” polypeptide, Nuc, and/or LukG antigen(s).

Preferably, the immunogenic composition of the present invention,comprising at least one S. aureus antigen or the immunotherapeuticcomposition comprising an antibody, preferably a polyclonal antibody,raised against said at least one S. aureus antigen, further comprises atleast one pharmaceutically acceptable excipient. The term“pharmaceutically acceptable excipient” is defined herein as acomponent, or combination of components, that is compatible with thepharmaceutical composition, does not generate unwanted side effects inthe patient, and that is generally considered to be non-toxic. Apharmaceutically acceptable excipient is most commonly implicated infacilitating administration of the composition, increasing productshelf-life or efficacy, or improving the solubility or stability of thecomposition. In some cases, the excipient itself may also have atherapeutic effect. The choice of said one or more excipients mayfurthermore depend on the desired route of administration. In thecontext of the present invention, the pharmaceutically acceptableexcipient may notably comprise one or more diluents, adjuvants,antioxidants, preservatives, buffers and solubilizing agents. As anon-limiting example, the pharmaceutically acceptable excipient maycomprise water, saline, phosphate buffered saline, sugars such assucrose or dextrose, glycerol, ethanol, propylene glycol, polysorbate80, poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castoroil (e.g. Cremophor EL), poloxamer 407 and 188, fat emulsions, lipids,PEGylated phospholipids, polymer matrices, biocompatible polymers,lipospheres, vesicles, liposomes, cornstarch, gelatin, lactose, sucrose,microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate,calcium sulfate, sodium chloride, alginic acid, croscarmellose sodium,sodium starch glycolate, and combinations thereof.

Methods for preparing immunogenic compositions which contain antigens(i.e., polypeptides) or immunotherapeutic compositions which compriseantibodies as active ingredients are furthermore well-known in the art.Formulations can include those suitable for nasal, topical, oral(including buccal and sublingual) and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form may vary depending upon thesubject and/or the particular mode of administration. The amount ofactive ingredient, which can be combined with a carrier material toproduce a single dosage form, will generally be that amount of thecompound that produces a therapeutic effect. Typically, suchcompositions are prepared as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active ingredient is often mixedwith excipients, such as one or more of those listed above.

As used herein, the term “S. aureus” refers to any strain of theStaphylococcus aureus species. The term encompasses laboratory strainsas well as clinical isolates. According to a preferred embodiment, S.aureus is resistant to one or more antibiotics, preferably a methicillinresistant S. aureus (MRSA). The term “methicillin-resistant” indicatesthe lack of susceptibility of a bacterial strain to the bactericidaleffects of methicillin. Resistance to methicillin is notably conferredby a mecA or mecC gene commonly located within a StaphylococcalChromosomal Cassette (SCC). MRSA strains are also natively resistant toall agents of the beta-lactam class, with the possible exception of theso called “fifth-generation cephalosporins,” with ceftaroline andceftobiprole being the first available agents. MRSA strains may furthercomprise resistance to additional antibiotics (e.g., glycopeptides,lipopeptides, mupirocin, quinolones, aminoglycosides, macrolides,rifampin, etc.). Alternatively, S. aureus may be methicillin-sensitiveS. aureus (MSSA). Methicillin-sensitive strains are susceptible to thebactericidal effects of methicillin and other beta-lactams nothydrolyzed by the class A beta-lactamases commonly observed in S. aureus(notably oxacillins, cloxacillin, nafcillin, cephalosporines,carbapenems, penicillins/beta-lactam inhibitor combinations) but maycomprise resistance to other antibiotics.

The expression “conferring protection against disease caused by S.aureus” as used herein refers to the prevention or the delay of theonset and/or establishment of a S. aureus associated disease orinfection. As a non-limiting example, said S. aureus disease orinfection may be a skin or soft tissue infection (SSTI), woundinfection, bacteremia, endocarditis, pneumonia, osteomyelitis, toxicshock syndrome, infective endocarditis, folliculitis, furuncle,carbuncle, impetigo, bullous impetigo, cellulitis, botryomyosis, scaldedskin syndrome, central nervous system infection, infective andinflammatory eye disease, osteomyelitis or other infections of joints orbones, respiratory tract infection, urinary tract infection, septicarthritis, septicemia, or gangrene. In particular, said S. aureusassociated disease or infection may be associated with the presence of aforeign device or implant in the subject, as described herein.

The “patient” or “subject” may be as any human individual, regardless oftheir age. Specifically, the subject may be an adult or child. The term“adult” refers herein to an individual of at least 16 years of age. Theterm “child” comprises infants from 0-1 years of age and children from1-8 years of age, 8-12 years of age, and 12-16 years of age. The term“child” further comprises neonatal infants from birth to 28 days of ageand post-neonatal infants from 28 to 364 days of age. The compositionmay be administered to an adult or a child, including a neonatal infant.The compositions of the invention are particularly advantageous for usein the prevention or treatment of S. aureus associated disease in asubject that will undergo or that has already undergone ahospitalization for any reason, more preferably a hospitalization forcardiac or orthopedic surgery, or a dialysis treatment (e.g., kidneydialysis). In a preferred embodiment, the subject bears a foreign deviceor implant. As a non-limiting example, said subject may bear one or moreof the following devices or implants: an intravenous catheter, avascular prosthesis, an intravascular stent, a cerebrospinal fluidshunt, a prosthetic heart valve, a urinary catheter, a joint prosthesis,an orthopedic fixation device, a cardiac pacemaker or defibrillator, aperitoneal dialysis catheter, an intrauterine device, a biliary tractstent, a catheter for insulin administration, dentures, breast implants,contact lenses, or any other foreign device or implant. Preferably, saidsubject has an osteoarticular device, preferably an osteoarticularimplant, more preferably a total or partial joint prosthesis, even morepreferably a total or partial hip, knee, shoulder, elbow, wrist, orankle replacement.

In a further aspect of the invention, the immunogenic orimmunotherapeutic composition according to any of the embodimentsprovided herein is for use in the treatment of an S. aureus infection ina subject. The term “treatment” refers to a process by which thesymptoms of an S. aureus infection are improved or completelyeliminated. Treatment is preferably performed by internal administrationof the immunogenic or immunotherapeutic composition as described hereinto a subject, in combination with one or more conventional therapies,such as antibiotic therapy used in the treatment or prevention of S.aureus infection, or concomitantly with implant replacement in the caseof implant failure due to S. aureus infection. Thus, according to aparticular embodiment, said immunogenic or immunotherapeutic compositionis for use in association with one or more antibiotics effective againsta S. aureus infection.

A further aspect of the present invention concerns a method of elicitingan immune response in a subject in need thereof, comprising providing oradministering the immunogenic composition described herein, for thepurpose of preventing or treating a S. aureus infection. The presentinvention further relates to a method of preventing and/or treating a S.aureus associated disease or infection, comprising administering animmunogenic or immunotherapeutic composition according to any of theembodiments as described herein in a subject in need thereof. Accordingto a particular embodiment, a method of conferring passive immunity to asubject in need thereof is provided herein, said method comprising thesteps of (1) generating an antibody preparation using an immunogeniccomposition comprising at least one S. aureus antigen, wherein saidantigen is a polypeptide having at least 80% identity with the SdrH-likepolypeptide of SEQ ID NO: 8, Nuc of SEQ ID NO: 4, and/or LukG of SEQ IDNO: 12; and (2) administering the immunotherapeutic preparation to saidsubject.

Preferably, said S. aureus is an antibiotic resistant S. aureus, morepreferably MRSA. Preferably, said subject bares a foreign device orimplant as described herein, more preferably said subject has anosteoarticular device, preferably an osteoarticular implant, morepreferably a total joint replacement prosthesis.

The present invention also comprises the use of the immunogenic orimmunotherapeutic composition according to the invention for themanufacture of a medicament for raising an immune response in a subject,preferably for the prevention and/or treatment of S. aureus associateddisease or infection.

The present invention also comprises the use of the immunogenic orimmunotherapeutic composition according to any of the embodimentsdescribed herein in the prevention and/or treatment of S. aureusassociated disease or infection.

According to a further aspect, the present invention relates to a kitcomprising the immunogenic or immunotherapeutic composition as providedherein and instructions for providing or administering the immunogenicor immunotherapeutic composition described herein to a subject.

As mentioned above, in view of the high cost and duration of clinicaltrials, an in vitro opsonophagocytosis (OPA) assay is commonly used as acorrelate of protection for assessing vaccine responses (Romero-Steineret al., 2006), as well as for evaluating antibody functionality, inparticular the ability of antibodies to promote uptake of S. aureus byprofessional phagocytes (Nanra et al., 2013; Fowler et al, 2013). Incontrast to previous methods, which notably use polymorphonuclearneutrophils, the inventors have developed the novel OPA assay providedherein for identifying target vaccine antigens capable of antibodiespromoting both uptake and killing of S. aureus.

Thus, according to a further aspect, the present invention relates to anin vitro method of identifying an antigen conferring protection againstdisease caused by S. aureus in a subject comprising:

-   -   a) incubating a solution comprising S. aureus with a solution        comprising antibodies raised against an S. aureus antigen,        preferably for one hour at 35° C., thereby obtaining a mixed        suspension,    -   b) contacting macrophages with the mixed suspension of step a),    -   c) removing the mixed suspension from macrophages, and    -   d) assessing internalization and killing of S. aureus bacteria        by said macrophages, wherein said antigen is considered to        confer protection against disease caused by S. aureus when said        antigen induces both increased internalization and killing of S.        aureus.

Steps a), b), c) and d) of the above method are necessarily performed inthe above-indicated order. Additional steps may furthermore be comprisedin the method, such as culturing or diluting a solution comprising S.aureus such that the bacteria are provided at a particular density (e.g.an optical density of 1), concentrating or diluting the solutioncomprising antibodies, and/or diluting the mixed solution (e.g. suchthat the macrophages may be contacted with bacteria having a particularmultiplicity of infection (MOI)), washing bacteria and/or macrophages,incubating macrophages, and the like.

While step a) is preferably performed for 1 hour at 35° C., incubationmay occur for one minute to 48 hours at a temperature ranging from 2° C.to 40° C. Similarly, while step b) is preferably performed for 1 hour at35° C., macrophages may be contacted with the mixed suspension for oneminute to 48 hours at a temperature ranging from 2° C. to 38° C.Preferably, macrophages are stored at 35° C. in an atmosphere of 5% CO₂.Preferably, the mixed suspension has a MOI comprised between 10:1 and25:1 (i.e., 10 to 25 bacteria per macrophage). The contact of themacrophage layer with S. aureus can be enhanced by centrifugation so thecontact between S. aureus and the macrophages is facilitated.Centrifugation may thus advantageously reduce the duration of step b).Said step of contacting allows a proportion of S. aureus bacteria to beinternalized, which may furthermore vary according to the composition ofthe solution comprising antibodies provided in step a). The removal ofthe mixed suspension in step c) may notably comprise one or more washingsteps (e.g., washing the macrophages with fresh culture media orphosphate buffered saline (PBS). Indeed, this advantageously improvesremoval of external S. aureus bacteria. Step c) may further comprise theaddition of a solution comprising antibiotics, preferably following theremoval of the mixed suspension. The addition of such a solutionadvantageously ensures that any remaining extracellular S. aureusbacteria are killed. Thus, S. aureus should be sensitive to theantibiotic used, while macrophages are preferably unaffected. As anon-limiting example, said antibiotic is gentamicin. Gentamicin maynotably be present at a concentration within the range of 50 μg/mL to100 μg/nnL, or at a concentration equal or superior to 100 μg/nnL. As anon-limiting example, said solution comprising antibiotics is a cellculture medium, such as DMEM. Said culture medium has preferably neverbeen used before (i.e., it is “fresh”).

Thus, according to a preferred embodiment, step c) comprises removingthe mixed suspension from macrophages and adding a solution supplementedwith antibiotics. More preferably, step c) comprises removing the mixedsuspension from macrophages and adding fresh medium supplemented withantibiotics, thus ensuring that extracellular S. aureus bacteria arekilled.

Internalization and killing of S. aureus bacteria in macrophages may bedetermined using methods known in the art.

As an example, internalization may be determined by quantifying bacteriaaccording to the level of fluorescence that is observed (e.g. directlyby measuring fluorescence of recombinant S. aureus bacteria expressing afluorescent protein, or indirectly by adding one or more staining agentsto fixed, permeabilized macrophages, for example BODIPY® FL Vancomycin(VMB), a fluorescent glycopeptide antibiotic that binds to the cell wallof gram positive bacteria, and measuring resulting fluorescence)according to methods known in the art (e.g. image analysis etc.).Internalization may notably be determined by comparing the level offluorescence present 3 h after step c) in macrophages treated with amixed suspension comprising antibodies raised against an antigen ofinterest versus control serum (e.g., raised against an antigen that isabsent in S. aureus, such as the GFP protein) is a using imageacquisition and analysis. As a particular example, image analysis may beused to determine e.g., the number of pixels positive for VMBfluorescence per macrophage in each condition. As a further example, thekilling of S. aureus bacteria in step d) is assessed by comparing thequantity of bacteria internalized in macrophages at 3 hours after stepc) with the quantity of bacteria internalized in macrophages at 6 hoursafter step c). Specifically, killing may be determined according to thelevel of VMB fluorescence that is observed according to the methodsdescribed herein at 3 h vs 6 h. Bacterial growth may be considered tooccur when increased fluorescence was measured at 6 h as compared to 3h. Bacterial lysis (i.e., killing) be considered to occur when adecrease in fluorescence was measured at 6 h as compared to 3 h. Suchchanges in fluorescence reflect the change in the amount ofintracellular peptidoglycan which is, associated with bacterialgrowth/lysis.

The macrophages used in the method may be any macrophage cell line orisolated macrophages. Preferably, said macrophages are cultured inmonolayers in classic culture conditions (i.e., in DMEM). Preferably,said macrophages are an immortalized macrophage cell line, morepreferably the J774.2 cell line.

DESCRIPTION OF THE FIGURES

FIG. 1 . S. aureus uptake mediated by immune sera

S. aureus uptake (3 h post-infection) at multiplicities of infections(MOIs) of 10:1 (left panels) et 25:1 (right panels), using immune seradiluted 1/1000 (upper panels) and 1/2000 (lower panels). Averagefluorescence areas values (488 nm excitation, 515 nm emission) arereported, normalized by anti-GFP antibody (value of 1). Standarddeviations were calculated from the values of fluorescence areas percell, before normalization relative to anti-GFP antibody fluorescence.Statistical significance was evaluated using Graphpad Prism on the rawdata. *P-value <0.05. **P-value <0.01. Proteins tested: Pbp2a (“A”),SspA (“B”), Sak (“C”), IsaA (“D”), GlpQ (“E”), Autolysin-like protein(“F”), Nuc (“G”), Hla (“H”), LukG (“I”), LukH (“J”), IsdA (“K”), IsdB(“M”), SdrD (as two partial polypeptides: “N” and “Nb”), ClfA (as twopartial polypeptides: “O” and “Ob”), MntC (“1”), SdrH-like polypeptide(“2”), Lip2 (“3”), putative protein (“4”), Atl (“5”), and hypotheticalprotein (“6”). Grey: Nuc (“G”), LukG (“I”) and SdrH-like polypeptide(“2”).

As shown, only five polypeptides are associated to a significantincrease of uptake in at least two different conditions: Nuc (“G”), Hla(“H”), LukG (“I”), IsdA (“K”), and SdrH-like polypeptide (“2”); notethat the values observed for dilutions 1/1000 et 1/2000 are similar,indicating the lack of threshold effect.

FIG. 2 . S. aureus killing

Killing of S. aureus (6 h post-infection) at MOls of 10:1 (left panels)and 25:1 (right panels), using immune sera diluted 1/1000 (upper panels)and 1/2000 (lower panels). The average fluorescence areas (excitation488 nm, emission 515nnn) of the reported protein at the 6 h time pointis normalized here to the value measured for the same protein at the 3 htime point (reference value of 1). Proteins tested are the same as thoselisted above (see legend of FIG. 1 ). Grey: Nuc (“G”), LukG (“I”), andSdrH-like polypeptide (“2”).

As shown, only three of the five antigens associated with a significantincrease in

S. aureus uptake are also associated with a killing of the bacteria, inall assay conditions: Nuc (“G”), LukG (“I”), and SdrH-like polypeptide(“2”).

FIG. 3 . Pictures of macrophages infected with S. aureus treated withanti-IsdB protein and anti-SdrH-like protein antisera at 6 hpost-infection (MOI 1:10, serum dilution 1/1000).

With anti-IsdB (“M”) protein antiserum (panel A), myriads of bacteria(white circles) can be seen filling up cytoplasmic space; areas of celllysis with release of extracellular bacteria can also be observed. Thiscontrasts with the picture obtained with anti-SdrH-like polypeptide(“2”) antiserum (panel B): individual bacteria (white circles) can beenumerated; the cytoskeleton structure is preserved and the nuclei areasare preserved. Similar observations were made with anti-Nuc (“G”)protein and anti-LukG (“I”) protein antisera (data not shown).

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. All subject-matter set forth or shown in the followingexamples and accompanying drawings is to be interpreted as illustrativeand not in a limiting sense. The following examples include anyalternatives, equivalents, and modifications that may be determined by aperson skilled in the art.

Example 1: Construction, Production and Purification of the S. aureusand Control Antigens Materials and Methods

Cloning of the Genes Coding the S. aureus Antigens of the Invention andS. aureus Control Antigens into an Expression Vector

The sequenced S. aureus strain Mu50 was used as a source of genomic DNA.DNA extraction was performed using a commercial kit (DNeasy Blood andTissue, Qiagen Hilden, Germany). S. aureus genes of interest wereamplified by polymerase chain reaction (PCR) using appropriate primers,designed with AmplifX.

Nucleotide sequences of S. aureus genes are notably as provided in SEQID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65,69, 73, and 79. Cloned DNA sequences are as provided in SEQ ID NOs: 2,6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74,75, 80, and 81. In particular, two different polypeptides were clonedfor the sdrD and clfA genes (SEQ ID NOs: 74 and 75 for sdrD and SEQ IDNOs: 80 and 81 for clfA). DNA was purified prior enzymatic restrictionwith Sail and Stul (Thermo Scientific, Waltham, USA), as was theexpression vector pET-6xHN-N (Clontech, Otsu, Japan) containing apoly-histidine tag. Restricted PCR products were then ligated into thevector. Resulting expression vectors of each gene were controlled byelectrophoretic migration prior to transformation into chemocompetentDH10131 Escherichia coli (Thermo Scientific). Transformed bacteria wereincubated 1 h at 35° C. in Luria-Bertani (LB) broth before being platedon LB agar with ampicillin (100 mg/L) and incubated overnight at 35° C.Isolated colonies were harvested and grown overnight in LB broth toamplify the clone. Vector DNA was then purified using a commercial kit(QlAprep Spin Miniprep, Qiagen). Sequencing was performed to validateeach inserted gene sequence. The pET-6xHN-GFPuv vector (Clontech) wasused for expressing the green fluorescent protein (GFP, SEQ ID NOs: 85and 86 for cloned DNA and amino acid sequences, respectively).

Antigen Production and Purification

Verified vectors were used to transform chemocompetent BL21 (DE3) E.coli cells (Thermo Scientific) following the same protocol as used forDH101β1 cells and isolated colonies similarly amplified. A 1/100dilution of the overnight culture was incubated at 35° C. until theculture reached an optical density (OD) of 0.5. A solution of IPTG (1 mMfinal) was then added to the bacteria to induce antigen production at35° C. until an OD of 1.2 was reached. Bacterial pellets obtained bycentrifugation were lysed and the Histidine tagged proteins purifiedusing a commercial kit (Proteus Metal Chelate, Generon, Slough, UK) andthe recombinant His-tagged proteins eluted using a 10 mM imidazolesolution. Eluted antigens were then concentrated using an AmiconUltra-15 column (Merck, Darmstadt, Germany). Polypeptide sequences ofsaid antigens are as provided in SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27,31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 76, and 82. Clonedpolypeptide sequences, all of which further comprise an N-terminal Histag, are as provided in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36,10, 44, 48, 52, 56, 60, 64, 68, 72, 77, 78, 83, and 84.

Antigen Characterization

Characterization of the purified antigens was performed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coloredwith a Coomassie solution to evaluate the size, integrity and purity ofthe recombinant antigen. The concentration of the purified antigensolutions was determined using Bradford's method.

Results

A total of 20 S. aureus antigens, evaluated as 22 differentpolypeptides, were cloned, expressed and purified (>95% of purity), withtotal amounts of purified protein ranging from 1 mg to 6 mg for eachrecombinant protein.

Four of these proteins are included in anti-S. aureus vaccinesundergoing pharmaceutical development and were used as control vaccineantigens: IsdB (described in Harro et al., 2010, Moustafa et al., 2012,Fowler et al., 2013), MntC (described in Salazar et al., 2014, Begier etal., 2017, Inoue et al., 2018), CIfA (described in Salazar et al., 2014,Begier et al., 2017, Inoue et al., 2018), and Hla alpha-toxin describedin Landrum et al., 2016).

Example 2: Production of Antibodies Against S. aureus Antigens Materialsand Methods

Antibodies targeting S. aureus polypeptides and control antigens wereobtained by immunization of specific pathogen free BALB/cJRj mice,specifically documented to have originated in a S. aureus-freeenvironment. Mice were received when they were nine weeks-old and wereacclimated one week prior to immunization. Groups of six mice perantigen were injected with a first dose of 20 μg of purified antigenwith Freund's complete adjuvant, followed by two more injections at 21and 42 days with 20 μg of antigen with Freund's incomplete adjuvant.Mice were sacrificed and the sera collected 50 days after the firstinjection. A first control group (n=6) was injected with GFP(non-relevant non-S. aureus antigen). A second control group (n=6) wasinjected with Phosphate Buffer Solution (PBS) to control for adjuvantimmunogenicity.

The titer and specificity of each immune serum were verified bywestern-blot using purified proteins.

Results

Each serum was evaluated with serial dilutions down to a titer of60,000. All serum sample tested showed a positive specific band at thisconcentration, showing the effective immunization of all animals. Serafrom the six mice immunized with the same antigen were pooled to obtaina single immune serum stock for each of the 22 S. aureus polypeptidesand for GFP.

Example 3: In vitro Evaluation of Immune Sera in a Macrophage-Based OPAAssay Materials and Methods

S. aureus Strains

Experiments were performed with USA300 and its spA⁻ derivative (FrédéricLaurent, Lyon).

Macrophage Culture

Cellular assays were performed on the murine BALB/c immortalizedmacrophage cell line J774.2 (European Collection of Authenticated CellLines, Porton Down, UK). Macrophages were cultured in Dulbecco'sModified Eagle Medium (DMEM) complemented with 10% fetal bovine serum at35° C. under a 5% CO₂ atmosphere. Cells were suspended in complementedDMEM, titrated, and seeded in culture plates 24 h prior to the assay.

Macrophage-Based Assay

An 18 h S. aureus culture in brain-heart infusion (BHI) broth wasdiluted 1/100 in fresh medium and incubated at 37° C. until the culturereached an OD of 1. Immune sera diluted 1/1000 or 1/2000 were added andallowed to bind to the bacterial surface for 1 h at 35° C. Serum-treatedbacteria were then added to the titrated J774.2 cell monolayers atmultiplicities of infection (MOI) of 10:1 (10 bacteria per cell) and25:1 (25 bacteria per cell). After incubation for 1 h at 35° C. under a5% CO₂ atmosphere, wells were emptied of medium and gently washed withPBS before adding fresh DMEM with gentamicin. At appropriate times (seebelow: bacterial uptake, 3 h post-infection; bacterial killing, 6 hpost-infection), J774.2 cells were washed with PBS, fixed with PFA 4%for 5 minutes, and then permeabilized with 0.1% Triton X100 for 5 min.Fixed cells were dyed for 30 min with Hoechst 33342 (Thermo Scientific),Phalloidin-ATTO 655 (Sigma-Aldrich, Saint-Louis, USA) and BODIPY® FLVancomycin (VMB) (Invitrogen, Carlsbad, USA), and were sealed usingglass coverslips. Images were acquired using a Leica SP8 confocalmicroscope and analyzed using ImageJ software (National Institute ofHealth, Bethesda, USA).

Evaluation of Bacterial Uptake and Killing

The uptake of serum-treated bacteria was evaluated at 3 h post-infectionby comparing the number of pixels with VMB fluorescence (bacterial cellwall quantification) per J774.2 cell for each antigen specific serum tothe number of pixels with VMB fluorescence per J774.2 cell for thenon-relevant control serum (anti-GFP). To evaluate the outcome ofinternalized bacteria, the area of VMB at 3 h post-infection and 6 hpost-infection were compared. Bacterial growth was called when anincrease of fluorescence was measured, reflecting an increase in theamount of intracellular peptidoglycan. Bacterial lysis (“killing”) wascalled when a decrease in the amount of intracellular peptidoglycan wasmeasured, reflecting a decrease in the amount of intracellularpeptidoglycan.

Results

The evaluation of bacterial uptake evaluated 3 hours after infection atMOI of 10:1 and 25:1 following S. aureus incubation with two antibodydilutions (1:1000, 1:2000) is presented here, featuring an anti-GFPcontrol serum in each experiment for normalization. Five antigens showmarkedly different behaviors with a significant increase in theinternalization of S. aureus bacteria in at least two conditions:proteins the SdrH-like polypeptide (“2”), Nuc (“G”), and LukG (“I”), Hla(“H”), and IsdA (“K”) (FIG. 1 ). Intracellular bacterial clearance andgrowth was evaluated by comparing the areas of VMB fluorescence 6 hoursafter infection to the areas of fluorescence observed 3 hours afterinfection. Among the five antigens previously shown to be associatedwith significant bacterial uptake, three were associated with bacterialkilling in all experimental conditions: the SdrH-like polypeptide (“2”),Nuc (“G”), and LukG (“I”) (FIG. 2 ). Noticeably, a number of proteinswith no significant effect on uptake were associated with bacterialgrowth enhancement (“facilitating” effect of immune sera) (see forexample, proteins Pbp2a (“A”) and Sak (“C”) in FIG. 2 , at a MOI of 25:1and with a serum dilution of 1/1000). Bacterial growth was particularlyintense with anti-IsdB protein sera and resulted in the destruction ofthe macrophage monolayer (FIG. 3 ), leading to underestimating the loadof intracellular bacteria (compare FIGS. 1 Et 2 with FIG. 3 ).

Example 4: Establishment of an in vivo Model of Systemic S. aureusInfection in Mice

Previous studies have shown that BALB/c mice are highly susceptible toblood-borne S. aureus infection, due to the inability of this mousestrain to limit bacterial growth in the kidneys (von Köckritz-Bliclwedeet al., 2008). However, as the course of infection may differ among S.aureus strains according to their virulence repertoire, we firstdetermined which dose of S. aureus USA300 led to non-lethal kidneyinfection.

Materials and Methods

S. aureus strains

Experiments were performed with S. aureus strain USA300.

Mice

Female BALB/c mice were purchased from Janvier Labs (Le Genest SaintIsle, France). Mice were received when they were six weeks-old and wereacclimatized one week prior to immunization. Animal experiments wereperformed according to institutional and national ethical guidelines(Agreement APAFIS #26827).

Mouse Model of Systemic S. aureus Infection

Mice were anaesthetized by intraperitoneal administration ofketamine/xylazine (50/10 mg/kg) and were inoculated with 10⁹, 10⁷ or 10⁵CFU of USA300 by retro-orbital sinus injection under a volume of 1004.Mice were euthanized 3 hours and 24 hours after infection. Spleen andkidneys were harvested, homogenized, and serial dilutions were plated onMueller Hinton 2 agar plates. CFUs were enumerated after 24 hours ofincubation at 37° C. (minimal detection limit: 2.69 log₁₀ CFU perorgan).

Results

The dose of 10⁹ CFU caused the death of 100% of animals before the 24 hpost-challenge time-point while 10⁵ CFU did not allow the establishmentof infection in the kidneys (no bacteria detected at 3 h and 24 hpost-challenge) (Table 2). Thus, 10⁷ CFU was the only dose to benon-lethal and to be associated with the infection of kidneys. Asexpected, bacterial burden was similar at 3 h and 24 h post-challenge inthe spleen, suggesting infection control, while bacterial growth wasdramatically increased in the kidneys (CFU differential of ca 4 log₁₀between 3 h and 24 h post-challenge) (Table 2).

TABLE 2 CFU counts at 3 h and 24 h post-challenge in non-immunizedanimals. Mean number of CFUs per organ, in log₁₀ª Spleen Kidneys USA300dose 3 h 24 h 3 h 24 h 10⁹ CFU 6.70 †^(b) 6.68 †^(b) 10⁷ CFU 5.71 5.582.69 6.08 10⁵ CFU 3.66 ND ND ND ªGroups of four animals per organ and ateach time-point. ^(b)All animals died before the 24 h post-challengetime-point. ^(c)Not detectable (minimal detection limit, 2.69 log₁₀CFU).

Example 5: Evaluation of the Protective Effect of the SdrH-likePolypeptide Versus Negative Control in a Mouse Model of Systemic S.aureus Infection

BALB/c mice have been shown to be able to control S. aureus infection bydeveloping a strong Th2 response (Nippe et al., 2011). We previouslyshowed in the OPA assay that sera directed against the SdrH-likepolypeptide, Nuc, or LukG enhanced the killing of S. aureus byphagocytes (see Example 3). We therefore studied whether the vaccinationof BALB/c mice with one of these three antigens, the SdrH-likepolypeptide (“SdrH-like”), may allow for an improved control of kidneyinfection in this model of systemic infection.

Materials and Methods Production and Purification of SdrH-like,Adjuvants

SdrH-like was produced and purified as described in Example 1. Adjuvants(aluminum hydroxide gel (Alhydrogel®) and aluminum phosphate gel(Adju-Phos®); InVivoGen, CA, USA) were used according the manufacturer'srecommendations.

Vaccination Protocol and End-Point Analysis

Mice were immunized intramuscularly once a week for 3 weeks with 10 μgof purified SdrH-like (5 μg with Aluminum hydroxide gel (right thigh;volume: 50 μL) and 5 μg with Aluminum phosphate gel (left thigh, volume:50 μL)); mice received the same quantity of adjuvants alone as anegative control.

Mice (groups of six mice per time point) were inoculated two weeks afterthe third immunization with a dose of 10⁷ CFU of USA300; the protocolwas otherwise as described in Example 4.

Results

As shown in Table 3, the bacterial load at 24 h post-challenge wasreduced by 0.53 log₁₀ CFU in mice vaccinated with SdrH-like versuscontrol mice. Although, kidney infection was not controlled invaccinated mice, bacterial growth was substantially reduced (+1.53 log₁₀CFU between 3 h and 24 h post-challenge versus+2.06 log10 CFU forcontrol mice). As expected, vaccination had a minimal impact on spleeninfection.

TABLE 3 CFU counts at 3 h and 24 h post-challenge in animals immunizedwith SdrH- like versus negative control. Mean number of CFUs per organ,in log₁₀ª Time post- Negative control^(b) SdrH-like challenge SpleenKidneys Spleen Kidneys  3 h 5.31 3.20 5.25 3.15 24 h 4.05 5.26 4.33 4.68 3 h-24 h Δ −1.26 +2.06 −0.92 +1.53 ªGroups of six animals per organ andat each time-point. ^(b)Adjuvants alone.

Example 6: Evaluation of the protective effect of SdrH-like versusstaphylokinase and MntC in a mouse model of systemic S. aureus infection

SdrH-like was then compared to staphylokinase and MntC. The firstcomparator, staphylokinase, was chosen because sera directed againstthis protein were paradoxically shown to favor the intracellular growthof S. aureus in the OPA assay (see Sak, “C”, in FIGS. 1 and 2 ). Thesecond comparator, MntC, was chosen because it has been shown to be apromising vaccine candidate in various animal models (Anderson et al.,2012), while it was revealed to be inferior to SdrH-like in the OPAassay (see MntC, “1”, in FIGS. 1 and 2 ).

Three infectious doses were tested: 10′, 3x10 6 and 10 6 CFU.

Materials and Methods

SdrH-like, staphylokinase and MntC were produced and purified asdescribed in Example 1. Vaccination protocol and end-point analysis wereas described in Example 5, except that protective effect was evaluatedusing three doses: 10⁷, 3×10⁶ and 10⁶ CFU of USA300.

Results

The course of infection in kidneys clearly differed in the micevaccinated with SdrH-like as compared to those vaccinated withstaphylokinase (Table 4); regardless of the dose of USA300, SdrH-likereduced the bacterial load in kidneys by ca 0.80 log₁₀ CFU as comparedto staphylokinase (Table 5).

A similar CFU reduction (0.95 log₁₀ CFU) was observed with MntC comparedto staphylokinase at the lowest dose, i.e., 10⁶ CFU (Table 5); however,the difference was much less at 10⁷ and 3×10⁶ CFU (reduction of only0.30 to 0.39 log₁₀ CFU; Table 5).

Consistent with the above results, SdrH-like appeared to have a strongereffect than MntC on kidney infection at the two highest doses, i.e., 10⁷and 3×10⁶ CFU (reduction of 0.45 and 0.47 log₁₀ CFU, respectively; Table5), while similar results were found at 10⁶ CFU. Thus, the bacterialkinetics observed in the kidneys after vaccination with SdrH-like,staphylokinase and MntC paralleled the bacterial kinetics observed withthese three antigens in the OPA assay.

As expected, vaccination with each of these three antigens had a minimalimpact on spleen infection.

TABLE 4 CFU counts at 3 h and 24 h post-challenge in animals immunizedwith SdrH-like versus staphylokinase and MntC. Mean number of CFUs perorgan, in log₁₀ ^(a) USA300 Time post- Staphylokinase MntC SdrH-likedose challenge Spleen Kidneys Spleen Kidneys Spleen Kidneys 10⁷ CFU 3 h5.28 3.00 5.09 3.20 5.24 3.55 24 h  4.41 4.77 4.55 4.58 4.27 4.48  3h-24 h Δ^(b) −0.87 +1.77 −0.54 +1.38 −0.97 +0.93 3 × 10⁶ CFU 3 h 4.972.69 4.91 2.85 4.93 2.85 24 h  3.79 4.46 3.90 4.32 4.08 3.85 3 h-24 h Δ−1.18 +1.77 −1.01 +1.47 −0.85 +1.00 10⁶ CFU 3 h 4.43 2.69 4.23 2.85 4.522.69 24 h  3.77 3.90 3.76 3.11 3.61 3.09 3 h-24 h Δ −0.66 +1.21 −0.47+0.26 −0.91 +0.40 ^(a)Groups of six animals per organ and at eachtime-point. ^(b)Bacterial growth between 3 h and 24 h is indicated by“+”, bacterial killing by “−”.

TABLE 5 Pairwise comparison of 3 h-24 h CFU differentials in thekidneys. 3 h-24 h CFU differentials, in log₁₀ (difference)^(a) USA300SdrH-like vs SdrH-like vs MntC vs dose staphylokinase MntCstaphylokinase      10⁷ CFU +0.93 vs +1.77 +0.93 vs +1.38 +1.38 vs 1.77(−0.84) (-0.45) (-0.39) 3 × 10⁶ CFU +1.0 vs +1.77 +1.0 vs +1.47 +1.47 vs1.77 (−0.77) (-0.47) (-0.30)      10⁶ CFU +0.40 vs 1.21 +0.40 vs +0.26+0.26 vs 1.21 (−0.81) (+0.14) (−0.95) ªIn bold, differences ≥ 0.5 log₁₀CFU.

Conclusions

The binding of specific antibodies to S. aureus can be beneficial to thehost, as they may inhibit physiological functions of extracellularantigens, increase the uptake by immune cells, facilitate phagocytosis,and/or improve bacterial targeting to phagolysosomal compartments. Moreparticularly, antibodies against S. aureus antigens may inhibitbacterial defense mechanisms targeting the bacterium to a favorableintracellular microenvironment, enhance the immune response byincreasing the processing of the bacterium for antigen presentation, andfoster bacterial clearance. However, certain antibodies have deleteriouseffects, enhancing bacterial virulence by inhibiting the function ofdeterminants that are adequately recognized by the immune system andwhich participate in the control of the infection by the host. Thehumoral response generated by a vaccine candidate should preferablyincrease bacterial uptake for optimal antigen presentation and enhanceintracellular bacterial lysis.

Sera directed against the SdrH-like polypeptide, Nuc, or LukG weresurprisingly shown to both promote the internalization of S. aureus bymacrophages and enhance the intracellular clearance of S. aureusfollowing phagocytosis.

It is noteworthy that none of the antisera raised against the candidatevaccine proteins Hla, MntC, and CIfA previously developed and shown tobe ineffective in clinical trials combined the two properties reportedhere. Moreover, the IsdB vaccine candidate showed to worsen the outcomeof vaccinated patients was proven to be deleterious in the macrophageassay reported here, with acute destruction of the macrophage layerfollowing enhanced internalization. These results further confirm thepertinence of the novel macrophage based in vitro assay provided hereinin identifying antigens conferring protection against disease caused byS. aureus in a subject.

In addition, the results of the macrophage based in vitro assay wereconfirmed in vivo in a systemic model of S. aureus infection usingBALB/c mice, which are highly susceptible to S. aureus due to theirinability to limit bacterial growth in the kidneys. Indeed, of the threeantigens evaluated in this model, the SdrH-like polypeptide showed thestrongest inhibitory effect on bacterial growth of S. aureus in thekidneys overall, followed by MntC, in-line with kinetics observed in themacrophage assay.

REFERENCES

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1-14. (canceled)
 15. An immunogenic composition comprising at least oneStaphylococcus aureus antigen, wherein said antigen is a polypeptidehaving at least 80% identity with the SdrH-like polypeptide of SEQ IDNO: 8, Nuc of SEQ ID NO: 4, or LukG of SEQ ID NO:
 12. 16. Theimmunogenic composition of claim 15, comprising an antigen having atleast 80% identity with the SdrH-like polypeptide of SEQ ID NO: 8 and anantigen having at least 80% identity with LukG of SEQ ID NO:
 12. 17. Theimmunogenic composition of claim 15, wherein said composition comprisestwo or more of said S. aureus antigens in the form of separatepolypeptides or in the form of one or more fusion polypeptides or bothin the form of separate polypeptide(s) and fusion polypeptide(s). 18.The immunogenic composition of claim 15, further comprising apharmaceutically acceptable excipient.
 19. A method for conferringprotection against a disease caused by S. aureus in a subject in needthereof, said method comprising the administration to the patient of theimmunogenic composition of claim 15, as a vaccine.
 20. A method topromote uptake and killing of S. aureus by phagocytes in a subject inneed thereof, said method comprising the administration of animmunotherapeutic composition comprising a polyclonal antibody whichselectively binds to at least one antigen a polypeptide having at least80% identity with the SdrH-like polypeptide of SEQ ID NO: 8, Nuc of SEQID NO: 4, or LukG of SEQ ID NO: 12 .
 21. The method of claim 20, whereinsaid immunotherapeutic composition further comprises a pharmaceuticallyacceptable excipient.
 22. The method of claim 20, wherein saidimmunotherapeutic composition is used as a passive immunotherapyconferring protection against a disease caused by S. aureus in saidsubject.
 23. The method of claim 19, wherein said S. aureus is amethicillin-resistant S. aureus (MRSA) or a methicillin-susceptible S.aureus (MSSA).
 24. The method of claim 19, wherein said subject has anosteoarticular device.
 25. The method of claim 19, wherein said subjecthas an osteoarticular implant.
 26. The method of claim 19, wherein saidsubject has a total joint replacement prosthesis.
 27. The immunogeniccomposition of claim 15, further containing one or more antibiotics thatare effective against a S. aureus infection.
 28. An in vitro method foridentifying an antigen conferring protection against disease caused byS. aureus in a subject, said method comprising: a) incubating a solutioncomprising S. aureus with a solution comprising antibodies raisedagainst an S. aureus antigen, thereby obtaining a mixed suspension, b)contacting macrophages with the mixed suspension of step a), c) removingthe mixed suspension from macrophages and adding fresh mediumsupplemented with antibiotics to kill extracellular S. aureus bacteria,and d) assessing internalization and killing of S. aureus bacteria bysaid macrophages, wherein said antigen is considered to conferprotection against disease caused by S. aureus when said antigen inducesboth increased internalization and killing of S. aureus.
 29. The methodof claim 28, wherein step a) is performed for one hour at 35° C.
 30. Themethod of claim 28, wherein said macrophages are an immortalizedmacrophage cell line.
 31. The method of claim 28, wherein saidmacrophages are the J774.2 cell line.
 32. The method of claim 28,wherein the killing of S. aureus bacteria in step d) is assessed bycomparing the quantity of bacteria internalized in macrophages 3 hoursafter step c) with the quantity of bacteria internalized in macrophages6 hours after step c).
 33. The method of claim 20, wherein said S.aureus is a methicillin-resistant S. aureus (MRSA) or amethicillin-susceptible S. aureus (MSSA).
 34. The method of claim 20,wherein said subject has an osteoarticular device.